Circulating RAS/RAF and DNA-Repair Gene Mutations Associates with High-Risk/Treatment Resistance in Primary Refractory Multiple Myeloma

Background Multiple myeloma (MM) is an incurable plasma cell malignancy with a median overall survival (OS) of 5 years in newly diagnosed (ND) patients. Real-world data reveals that 23% of transplant eligible newly diagnosed MM patients relapse within 12 months of starting first-line bortezomib (1LB) based therapy and of these ~50% will fail secondary therapy and die within 18 months of diagnosis. It is currently impossible to identify these high-risk patients and genomic studies could potentially inform alternative secondary therapeutic options. We propose that circulating tumour DNA (ctDNA) analysis could provide a more holistic approach to gain an insight into the genomics of high-risk patients than bone marrow (BM) tumour DNA analysis in this genetically heterogenous multi-site malignancy.

Methods A total of 186 paired peripheral blood plasma and BM samples were obtained at baseline, cycle 3 day 1 (C3D1), end of study (EOS) and/or relapse, whichever appeared earlier, from a Phase II single arm study of carfilzomib-thalidomide-dexamethasone (KTd) in 50 transplant-eligible ND MM patients who were refractory or had a suboptimal response to 1LB (Australasian Leukaemia and Lymphoma Group MM17 trial). Somatic variants were identified with ultra-sensitive targeted amplicon sequencing of BM and plasma for 22-genes known to be mutated in MM. The mutational profiles were combined with the International Staging System (ISS), SKY92 MMProfiler^TM risk profile - standard-risk (SR) or high-risk (HR) or EuroFlow minimal residual disease (MRD) status and correlated to progression-free survival (PFS). Longitudinal monitoring of ctDNA variant allele frequency was also plotted to observe alterations in ctDNA kinetics at relapse compared to baseline and C3D1.

Results We performed a comparison of the available baseline ctDNA and BM mutational profiles between patients who did relapse (R) (n=18 ctDNA and n=10 BM) or did not relapse (NR) (n=29 ctDNA and n=21 BM). Utilising data derived from all variants identified, there were no significant differences in the relative proportions of specific mutations between R and NR in the BM samples whereas there were significant ctDNA differences between R and NR for both BRAF (p=0.02) and TP53 (p=0.06) mutations (chi-square test). We next performed a comparison of only those ctDNA variants with variant allelic frequencies (VAF) >1%, a threshold known to have a prognostic impact, and identified a statistically significant difference in mutational proportions between R and NR for RAS/RAF (22.2% vs 3.4%, p=0.04) and DNA damage repair genes (DDR) (ATM/ATR/TP53) (55.5% vs 20.6%, p=0.01). Subsequently, we combined ctDNA RAS/RAF and/or DDR mutational detection (>1% VAF, referred to as ctDNA+) with risk status as defined by ISS, SKY92 or MRD. Patients in ISS 2+3/ctDNA+ (compared to ISS 2+3/ctDNA-, ISS 1/ctDNA+, ISS 1/ctDNA-, Figure 1A); SKY92 HR/ctDNA+ (compared to SKY92 HR/ctDNA- and SKY92 SR/ctDNA+ or SKY92 SR/ctDNA-) and MRD+/ctDNA+ (compared to MRD+/ctDNA- and MRD-/ctDNA+ or MRD-/ctDNA-) all had significantly shorter PFS (p<0.01; p<0.01 and p<0.05; respectively, Log-rank tests). Importantly, patients that were ctDNA+ also had a shorter PFS on KTd compared to ctDNA- (p<0.01, 28.4 months for ctDNA+ vs not reached for ctDNA-, Log-rank test, Figure 1B). Conversely, parallel combined analyses with BM mutations revealed no significant prognostic differences. Longitudinal monitoring of ctDNA demonstrated that in 87.5% of patients, 1 or more of the dominant mutations present at the time of relapse were already present at the start of KTd salvage therapy.

Conclusions These data demonstrate that combining ctDNA mutational profiling with available platforms for MM risk-stratification significantly enhances the identification of high-risk patients. Moreover, the impact in this context of RAS/RAF and DDR mutations provides a rationale for the design and implementation of novel treatment paradigms utilising RAS/RAF inhibitors or DNA-repair modulators in these high-risk MM patients.

Reynolds:Novartis: Current equity holder in publicly-traded company; Abbvie: Research Funding; Alcon: Current equity holder in publicly-traded company. Quach:Karyopharm: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: receipt of free drug for investigator-initiated study, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: leadership or fiduciary role , Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees, Other: receipt of free drug for investigator-initiated study, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; CSL: Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: leadership or fiduciary role, receipt of free drug for investigator-initiated study , Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Antengene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: leadership or fiduciary role, receipt of free drug for investigator-initiated study , Research Funding. Horvath:sanofi: Research Funding; bms: Research Funding. Durie:Celgene/BMS: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Spencer:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria; Haemalogix: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria.